Separation of formed and fluid blood components



Feb. 4, 1958 EQ J. coHN SEPARATION OF' FORMED AND FLUID BLOOD COMPONENTS Feb. 4, 1958 E. .1. coHN 2,822,315

SEPARATION OF FORMED AND FLUID BLOOD COMPONENTS Filed April A12, 1952 s sheets-sheet 2 62u' .fm y 4MM Feb. 4, 1958 E. J. coHN 25822315 SEPARATION 0F FORMED AND FLUID BLOOD COMPONENTS Filed April 12. 1952 s sheets-sheet s as v //f\ 90 alaaf L s'PARAnoN or roman AND FLUID Loon coMroNENrs Edwin I. Cohn, Cambridge, Mass.; Rebekah R. Cohn, Charles A. Coolidge, and Eustace Seligman, executors of said Edwin I. Cohn, deceased, assignors to Protein Foundation, Incorporated, Cambridge, Maas., a nonprofit corporation Appuauon Aprit 12, 1952, serial N0. 281,989 somme. (ci. ns1-14) This invention relates to the treatment of animal body thereof in a closed system having a continuous feed.

The system is bio-mechanical in the sense that it combines automatic treatments, in series or station sequence, and of physical, physico-chemical and/or chemical nature, of a material such as blood fed continuously from a source, either a point of Veni-puncture or a reservoir of fresh or out-dated blood, looking towards separation of valuable therapeutic products, not only in stable states but in stable states more nearly identical with those in which the products exist in the body.

The obvious complexity ofthe role of blood in the human body is refiected in a nearly equal complexity of the relationships inter se of the components oi the blood. The close proximity of living cells of differing life expectancies, enzymes. their substrates, proteins, hormones, metals, metal-carriers, etc. is responsible for a state of ux which, though controlled in the body by equilibria and regenerating mechanisms, rampages when no longer controlled by such mechanisms.

Upon blood-letting, clotting of the blood.is the most apparent of the immediate transitions, but the other transitions in whole blood are legend, many still to be comprehended.

Extensive blood studies have revealed that a substantial number, and eventually it may be that essentially all, of the components of the blood can be preserved substantially in their native states if separation from deteriorating adjuncts, and concentration and purification to stable states can be achieved in the shortest possible periods of time under conditions best known to minimize deterioration, for example in the case of proteins, under conditions of low temperature which are now known to I minimize denaturation.

Nonetheless, the recovery of an individual or only a few components without attention to all the components is a gross waste of blood, the available supply of which is by no means inexhaustible.

This invention, therefore, is directed to the provision of bio-chemical methods for carrying out with a rapidity unattainable in prior apparatus, themost improved methods of blood component separation, utilizing as certain elements of the system improved centrifuges as hereinafter described, whichhave the additional value, by reason `of their closed nature, of permitting sterility maintenance throughout processing.

Since the method of this invention best utilizes continuous feed centrifuges, suitable centrifuges of this type are shown in the accompanying drawings and will now be described.

Fig. 2 is a sectional view of a centrifuge of particular use in carrying 'out that step in the method of this inven tion which accomplishes the separation of certain of the c ellular components of the blood, namely, the redcells and the white cells, from the plasma, while Fig. 3 is a similar cross-sectional view of a modifiedI form of centrifuge which is used at subsequent stations in the carried out therein.

liquids such as blood, for the separation of components Patented Feb. 4, 1958 ice . 2 method for the purpose of recovering various individual proteins from the plasma.

'I'he structural features of the centrifuges of Figs. 2 and 3 .are claimed in an application filed simultaneously herewith, as well as certain processing methods which may be will be briefly set forth herein. v

Referring to Pig. 2, the centrifuge bowl is .made up of two parts. The first of `these is a closed top conical side wall, internally silicone-coated glass container 80 having a circular restricted bottom opening. The second part comprises a lower tubular member 81 also of internally silicone-coated `glass which is inserted upwardly in the bottom opening of the bowl 80. The lower portions of the part 81 include an enlarged diameter section 82 and a section of intermediate diameter 83. The upper portion of the part 81 includes a section 84 which acts as a retarder and has a series of circumferentially spaced apertures 85 for permitting egress of liquid from the upper bowl 80 into the lower part 8l. l

The parts 81 and 82 are provided'with ground 'intertting surfaces at 86.

A central stationary feed tube 87 extends upwardly through the part 81 and has at its top a convex surfaced cap 88 having outlet apertures below the top of its convexity so that the tube 87 may lightly touch the top wall tube 87, together with a surrounding sleeve 90, are both seated in a fixture 91 which fits downwardly into a bevelled aperture 92 in a bottom plate 93.

The fixture 91 also supports a second tube 94 so that the combined fixture provides three separate inlets to the bowl chamber, the tube 87 being for blood, the sleeve 90 being connected to an inlet 95 for gas inow and the tube 94 being connected to an inlet 96 for ,introduction of reagents, if required.

The stationary plate 93 also carries fixed thereto an upstanding cylindrical glass bearing support 97 on top of which is seated a stainless steel annulus 98 with an inter-- posed soft gasket 99.

The base plate also supports a concentric baille 100 and upwardly dared separator 101. An outlet through the base plate is provided at 102 and at 103, one inside and one outside the separator 101.

Asealing surface with annulus 98 is provided at the bottom lapped surface of a compressed graphite ring 104 which bears upwardly against a bearing ring 105 against the yield of an undulating annular metallic spring 106 seated against the flange 107 o f an internal housing 10,8. A gasket 111 is interposed between ring 104 and housing 108. The housing 108 is screw-threaded into an exterior sleeve 109 which has connected thereto a curved annular rubber gasket 110 for receiving the lower portion of the glass element 82.

All the elements 104-110 rotate with the upper bowl by virtue of three circumferentially spaced connecting pins, one of which is shown at the left in Fig. 3 at 112, being threaded through a boss 113 on the housing 109, thus attaching the lower portion of the assembly 104-110 for rotation with an inside upper sleeve 114 which engages the upper bowl 80 with an interposed rubber gasket 115. An outside pulley sleeve 116 is designed to receive the inside sleeve ll4 with frictional engagement, thus supporting the bowl 80 and its assembled parts for rotation. The sleeve 116 is supported in ball bearings and driven by a belt'(not shown) engaging the pulley groove 117.

ln order to hold the upper and lower parts of the assembly which can rotate relative to one another against vertical separation, bolts 120, one of which is shown at the right in Fig. 2, are provided having lost motion con- However, their structural features v be retained on the resin column.

nection with a groove 121 in the external housing sleeve 109. As shown', parts of the stationary frame may be provided ,with internal conduits 122 for the circulation of coolants.

In the Fig. l flow sheet, a centrifuge of the type shown in Fig. 2 is positioned at A and separate centrifuges of the type shown in Fig. 3 are supplied at the positions and 1n the sequence indicated by B. Fig. l shows a donors arm from which the blood is led through a cation exchange resin column. This resin may be a cross-linked vinyl copolymer having sulphonic or carboxylic acid groups as exchange centers, the spheroidal particles of -40 mesh. Particular efforts must be taken to free the resin of heavy metals where the recoveries are intended for therapeutic use. A typical resin used is available under the trade name Dowex S0.

From the resin column, the blood flows directly through a heat exchange column to reduce the temperature as quickly as possible to 0 to 4 C. and, thence, flows directly into a cell centrifuge, which may be externally refrigerated.

It has been found that platelets of the blood tend to The present explanation is that the platelets interact with calcium, with the other valence of the calcium being covered by bicarbonate. Loss of carbon dioxide permits the calcium of the blood and the platelets to be held by the resin. Essentially quantitative recovery of the platelets is obtained if, in addition to calcium normally in the blood, a few of the exchange sites on the resin have been saturated with calcium before the commencement of blood ow to attract non-calcium bound platelets of the blood. This can be accomplished in a batch operation equilibrating the resin for one hour with l0 volumes of a solution of 0.15 M sodium chloride and 0.0005 M calcium chloride.

Alternatively, saturation of a few of the exchange sites on the resin with zinc results in equally good yields. Magnesium, however, is ineffective. The platelets may be eluted from the resin, after displacement of the blood remaining in the resin bed, with reagents which compete with the platelets for the metal. Such reagents include neutral salt solutions such as acetate citrate or a sequestering agent, such as ethylenediaminetetraacetate, made isotonic with sodium chloride.

Prior to the commencement of the donation, the rotating cell centrifuge is primed with 100 cc. of a preservative solution having a density higher than that of the plasma and near that of the white cells but lower than that of the red cells. The solution may contain rouleauforming agents, such as amylose, dextrin or glutamylpeptide, and, if desired, certain of the cations other than calcium which may have been removed on the resin exchange column. Useful solutions are 14% lactose, 4%

amylopectin solution or a 14% lactose, 0.28% sodium chloride solution. V

The centrifugal force applied in the cell centrifuge may be of the order of X gravity at the inner cylinder. The red blood cells will pass into and traverse the high density solution. In this low centrifugal field, and with the short contact time which prevails, the white blood cells will pass down the inner rotating interface of the high density solution with the plasma. cells will pass through the apertures 85, pass through the space above the collector 101 and the white cells will be trapped in the section 82, the plasma continuing to ow down to the collector plate 93 and passing through the outlet 103. The dimensions of bowl and of the section 82 are such that at the end of the blood donation, the upper bowl 80 will contain substantially entirely red blood cells and high density solution. The section 82 will contain substantially entirely white cells and substantially all of the plasma will have ilowed through the outlet 103. When the rotating bowl is stopped, the high density solution and red blood cells will pass through the The plasma and white blood apertures 85 and downwardly inside the collector 101 and through the outlet 102, whereas the white blood cells may thereafter be washed from section 82 and follow the plasma through the outlet 103 into a separate collecting device..

The efliuent plasma, as it llows uninterruptedly from outlet 103, is fed directly to another centrifuge which may be of the simpler type shown in Fig. 3. The Fig. 3 centrifuge is similar to that shown in Fig. 2, but, since the dimensions of the bowl are different, the lower portion of the device is somewhat dierently proportioned.

The stationary central tube parts have the same three infeeds, including the parts 87--96, identical with the same parts shown in Fig. 2. The' same is true of the parts 109, 112-117, 120 and 121. The bowl 80a is, however, of much greater vertical dimension and has a slightly smaller bottom opening and, therefore, it has a longer retarder 84a. In this case, the lower part of the member 84a has an outwardly extending ange 130 which engages a lip 132 on the sleeve 133 which is screw-threaded to the external housing 109. The member corresponding to the internal sleeve 108a in Fig. 2 is dierently dimensioned, as are the smaller undulating spring 106a, sealing ring a and compressed graphite ring 104a, but, otherwise, these parts, as well as the collector plate parts 97a and 98a function in the same manner as the corresponding-parts in Fig. 2.

In this case, there is need of only one opening 102a in the base plate 93a inasmuch as the centrifuge is designed principally for the separation of proteins and n which a precipitate is left in the bowl 80a and soluble components pass downwardly through the apertures 85a and outlet 102a continuously after the bowl has been filled so that its internal rotating wall surface is at the radius of the apertures 85a.

Referring again to Fig. 1, the plasma efiluent from the cell centrifuge ows directly into a Pig. 3 type centrifuge which contains a suspension of barium sulphate or other absorbent for prothrombin and the prothrombinfree effluent then flows into a container having protein separation reagents, for example, the zinc reagent referred to in my co-pending application Serial No. 212,204, now Patent No. 2,770,616, i. e. 5 mM. of zinc per 500 ce. donation.

After suflicient stirring and retention in this container for reaching equilibrium, the reagent-containing plasma is pumped with nitrogen pressure, or otherwise carried, to a third centrifuge of the Fig. 3 type wherein the precipitate of plasma globulins is collected, the unprecipitated components of the plasma then owing to a cation resin exchange column for removing the zinc from the albumin solution referred to in my co-pendng application as S. P. P. S. In this case, a carboxylated resin has proven far superior to a sulphonated resin. The addition of sodium caprylate in a concentration of 4 mM. or of triethylenetetramine facilitates the removal of the zinc.

. The resin particle size may be 15G-200 mesh. The pH of the resin was 7.5 and the temperature 0 C. utilizing the resin commercially known as IRC-50."

The plasma globulins (PGP) remaining in the third centrifuge may be removed for further treatments in similar centrifuges for the separation of gamma globulins, beta lipoproteins, fibrinogen and other proteins, all in accordance with the chemical processes described in my co-pending application filed January 29, 1951.

The closed system afforded by the protein centrifuges of the type shown in Fig. 3 permits the use of gaseous reagents for changing solubility conditions. Thus, in extracting gamma globulins from PGP, it has been found that themcre treatment of an aqueous suspension of PGP with carbon dioxide at a pressure of 1 atmosphere lowers the pH suiciently, for example, to 5.5 so that the gamma globulins become differentially soluble with respect to fibrinogen and the, B1 lipoproteins.

As indicated in Fig. 1, therefore, the gamma globulins may be obtained as a precipitate from the carbon dioxide containing efiluent merely by displacing the carbon dioxide with an inert gas such as nitrogen.

The use of such gaseous reagents, as permitted by the centrifuges herein described, can be responsible for wide changes in pH withoutthe complications and increased solubilities resulting from changes in ionic strength accompanying the use of acid additions with subsequent neutralization with alkali.

As indicated in the drawing Fig. la, the system is equally applicable to the treatment of out-dated blood. Here, the citrate previously added for preservation purposes must initially be removed by passage through an anion exchange resin which replaces or precedes the calcium removal step of the direct donation. Alternatively, the elect of the citrate may be overcome by dilution of the citrate-containing plasma with water to a volume 2 to 4X the volume of the original plasma. In either case, the following steps remain the same.

It is understood that all of the tubing is formed from non-wettable plastics, such as Teflon or silicones, and that all the metallic and glass parts with which the bloo'd may come into contact are properly coated to provide nonwettable surfaces also. All the parts are internally sterilized by known autoclaving methods prior to use.

Certain of the aspects of this invention are claimed in my application Serial No. 281,988 filed simultaneously herewith.

I claim:

1. The method of quantitatively removing viable platelets from human blood comprising passing fresh human blood over a cation exchange resin having on at least some of its sites before the blood is passed therethrough, a metal selected from the group consisting of calcium and zinc to collect platelets on said resin and thereafter removing the platelets collected on the resin from the resin.

2. The method of quantitatively removing viable platelets from human blood comprising passing fresh human blood over a cation exchange resin having added calcium at at least some of the exchange sites to collect platelets on said resin before the blood is passed therethrough, and thereafter eluting the platelets collected on the resin from the resin.

3. The method of quantitatively removing viable platelets from human blood comprising passing fresh human blood over a cation exchange resin having added calcium at at least some of the exchange sites to collect platelets on said resin before the blood is passed therethrough, and thereafter eluting the platelets collected on the resin from the resin with a salt solution.

4. A method of quantitatively separating red cells and platelets from other components of human blood comprising passing fresh human blood over a cation exchange resin having on at least some of its sites before the blood is passed therethrough, a metal selected from the group consisting of calcium and zinc to collect platelets on said resin, llowing the plateletpoor blood through a closed system and centrifuging the blood as it ilows to the action of centrifugal force to divert red cells from the path of flow of the plasma, continuing the plasma ow away from the diverted red cells, and separately collecting the platelets by removing them from the resin, the diverted red cells and the red-cell-free plasma.

5. The method of treating non-clotting blood as it ows continuously from a source of supply which comprises centrifuging the non-clotting blood as it continuously ows in the form of a film across and ot a layer of a solution having a density less than the density of the red cells but greater than the density of the plasma and white cells to divert red cells from the path of ow of the plasma and white cells, continuously collecting the diverted red cells separated from the plasma owing off said layer and then centrifuging the plasma and white cells after they flow off said layer to separate vthe white cells from the plasma.

References Cited in the iile of this pa'tent Cohn: PB Report No. 77, 276, Feb. ll, 1942, pp. 1-14.

Callahan: Chem. and Met. Eng., June 1946, pp. lOl-103.

Cohn et al.: I. Am. Chem. Soc., vol. 72, 1950, pp. 465-474. v

Cohn et al.: Science, vol. 112, Oct. 20, 1950, pp. 450-1. 

5. THE METHOD OF TREATING NON-CLOTTING BLOOD AS IT FLOWS CONTINUOUSLY FROM A SOURCE OF SUPPLY WHICH COMPRISES CENTRIFUGING THE NON-CLOTTING BLOOD AS IT CONTINUOUSLY FLOWS IN THE FORM OF A FILM ACROSS AND OFF A LAYER OF A SOLUTION HAVING A DENSITY LESS THAN THE DENSITY OF THE RED CELLS BUT GREATER THAN THE DENSITY OF THE PLASMA AND WHITE CELLS TO DIVERT RED CELLS FROM THE PATH OF FLOW OF THE PLASMA AND WHITE CELLS, CONTINUOUSLY COLLECTING THE DIVERTED RED CELLS SEPARATED FROM THE PLASMA FLOWING OFF SAID LAYER AND THEN CENTRIFUGING THE PLASMA AND WHITE CELLS AFTER THEY FLOW OF SAID LAYER TO SEPARATE THE WHITE CELLS FROM THE PLASMA. 